Even though radioimmunoassay (RIA) developed in 1960 is applied to quantify trace levels of biomolecules (e.g., hormone, tumor markers) the use of isotope molecules tagged antigen or antibody has various problems related to stability of labeled biomarkers (e.g., short half-life, radiolysis) and safety (e.g., laboratory personnel, waste, the requirement of building special laboratory facilities).
In order to solve the problems occurring from RIA, enzyme immunoassay (EIA) methods capable of quantifying various biomarkers with an enzyme rather than radioactivity as the reporter label were developed with various detections. Sensitivity of EIA depends on the physical properties of applied detection method such as colorimeter, fluorescence, and chemiluminescence. EIA with chemiluminescence detection using luminol or 1,2-dioxetane is much more sensitive than other immunoassays including RIA. Also, the sensitivity of chemiluminescence EIA (CLEIA) is better than those of chemiluminescence immunoassay (CLIA) using acridinium ester- or Ruthenium chelate-labeled antigen or antibody. Thus, CLEIA is widely applied to quantify toxic biomolecules and drugs as well as to diagnose various diseases. However, applications of CLEIA commercialized in the current market aren't as wide as those of fluorescence EIA. This is because luminol and 1,2-dioxetane derivatives widely applied as a CL substrate under CLEIA only react with a specific label enzyme. For example, CLEIA using luminol is applied when luminol reacts with antigen or antibody labeled with horseradish peroxidase (HRP) for 15˜60 minutes. In the case of CLEIA using 1,2-dioxetane derivatives, maximum CL is measured when 1,2-dioxetane and antigen or antibody conjugated to alkaline phosphatase (ALP) are incubated for 5˜60 minutes.
It is well-known that peroxyoxalate chemiluminescence (POCL) detection is much more sensitive and selective than other CL detections. Unfortunately however, due to the instability of POCL reagents (e.g., bis(2,4-dinitrophenyl) oxalate (DNPO), bis(2,4,6-thrichlorophenyl) oxalate (TCPO)) in aqueous solution, it is difficult to apply POCL detection in CLEIA.
Recently, 1,1′-oxalyldiimidazole (ODI) derivatives' and 1,1′-oxalyldisodium benzoate (DOB) derivatives2 were synthesized as new POCL reagents. Trace levels of fluorescent biomolecules dissolved in aqueous solution were quantified using the new POCL reagents, based on the reaction mechanism shown in Scheme 1 and 2, even though they are also unstable in aqueous solution. This was possible because the reaction between H2O2 and ODI or ODB is faster than their decomposition in aqueous solution. Using the chemical and physical properties of the new POCL reagents, novel CLEIA was developed. The sensitivity of this CLEIA was better than that of currently applied CLEIA. Also, the applications of CLEIA using ODI or ODB derivatives were as wide as those of fluorescence EIA because low concentrations of antigen or antibody conjugated to various enzymes including HRP and ALP were quantified under the novel CLEIA.

